A PODXL open reading frame fragment was amplified by polymerase chain reaction (PCR) with KOD FX Neo (TOYOBO, Osaka, Japan), and the sequence of the PCR product was verified as wild‐type by sequencer. The purified DNA PCR product and the pMSCV plasmid (Riken Bioresource Center, Tsukuba, Japan) were ligated using an In‐Fusion HD Cloning Kit (Clontech Laboratories, Mountain View, CA, USA). Plat‐GP packaging cells were seeded at 70% confluence. Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA) was used to transfect the plasmid containing PODXL‐pMSCV and the packaging plasmid pVZV (Clontech Laboratories) into the Plat‐GP cells. After 24 h of incubation at 37°C, the complete medium was replaced. After a further 24 h incubation, cell culture supernatants were collected through a 0.45‐μm filter and stored at −80°C. Prior to transduction, A549 cells were seeded and incubated at 30% confluence. After replacement of the medium with D‐MEM containing 5 μg/mL polybrene (Nacalai Tesque, Kyoto, Japan), the purified virus in D‐MEM containing 5 μg/mL polybrene was added to the medium. The virus‐containing medium was discarded after 24 h incubation and the transduced cells were incubated with D‐MEM containing 10% FBS at 37°C for 24 h. Cells were selected for drug resistance in the presence of 5 μg/mL puromycin.
Polybrene
Manufactured by Nacalai Tesque
Sourced in Japan
Polybrene is a cationic polymer used to facilitate the binding and uptake of nucleic acids, such as DNA and RNA, during transfection experiments. It acts as a facilitator for the delivery of genetic materials into cells.
Automatically generated - may contain errors
Lab products found in correlation
Puromycin, by InvivoGen (7 mentions)
Pspax2, by Addgene (5 mentions)
Blasticidin s, by InvivoGen (5 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (4 mentions)
Lenti x concentrator, by Takara Bio (4 mentions)
Opti mem, by Thermo Fisher Scientific (4 mentions)
Fugene 6, by Promega (3 mentions)
Dulbecco modified eagle medium (dmem), by Nacalai Tesque (3 mentions)
Fugene hd transfection reagent, by Promega (3 mentions)
Pcmv vsv g, by Addgene (3 mentions)
Hygromycin, by Fujifilm (3 mentions)
Polyethyleneimine, by Polysciences (3 mentions)
Zeocin, by InvivoGen (3 mentions)
Puromycin, by Thermo Fisher Scientific (3 mentions)
Retro x concentrator, by Takara Bio (3 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (3 mentions)
Purefection reagent, by System Biosciences (3 mentions)
Virapower lentiviral packaging mix, by Thermo Fisher Scientific (2 mentions)
Polyethylenimine max, by Polysciences (2 mentions)
X tremegene 9 dna transfection reagent, by Roche (2 mentions)
52 protocols using polybrene
1
Overexpression of PODXL in A549 cells
2
Generating Lentiviral Vectors for Gene Overexpression
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The plasmids used in this study (pMRX-IP-GFP-LC3-RFP, pMRX-IP-WDR45-EGFP, and pMRX-IP-FLAG-NCOA4) were kindly gifted by N. Mizushima from the University of Tokyo. Each plasmid was transformed into E. coli DH5α (TaKaRa, 9057) and amplified. The plasmids were extracted using the QIAGEN Plasmid Kit (QIAGEN, 12125 or 12143) according to the manufacturer’s instructions, and the sequences were confirmed using the Sanger method. HEK293T cells (purchased from RIKEN BRC) were transiently transfected with each plasmid along with pCG-VSV-G and pCG-gag/pol (kindly gifted by T. Yasui, National Institutes of Biomedical Innovation, Health and Nutrition, Osaka, Japan) using the Lipofectamine 3000 reagent (Thermo Fisher, L300015). Three days after infection, the virus-containing supernatant was collected and concentrated by centrifugation (2330 × g, 20 min) using a dialysis filter (Amicon Ultra-15; Merck, UFC 910008). Fibroblasts were incubated with the collected virus-containing medium and 4 μg/ml of polybrene (Nacalai Tesque, 12996-81) for 8 h and 2 μg/ml of polybrene for 1 day. Uninfected cells were removed using 1 ng/μl of puromycin (InvivoGen: ant-pr-1).26 (link) Single clones were isolated using the limiting dilution technique.
3
Lentiviral shRNAmir Construct Transduction
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PLKO.1 lentiviral shRNAmir constructs were obtained from Thermo Fisher Scientific (Waltham, MA, USA; HMGA2 shRNA, RHS4533; negative control shRNA, RHS 4080). The constructs were co-transfected with the packaging construct (psPAX2) and the VSV-G envelope expression plasmid (pMD2.G), both purchased from Addgene (Cambridge, MA, USA), into 293FT cells using FuGENE 6 (Promega, Madison, WI, USA). For infection, cells were incubated with lentiviral particles and 4 µg/ml polybrene (Nacalai Tesque, Kyoto, Japan), and then selected with puromycin.
4
Lentivirus-Mediated Stable Gene Expression
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For lentivirus-mediated stable introduction of sfGFP-DARPin-LA6 and NLSSV40-sfCherry-NLSMyc, we followed the methods described previously (Liu et al., 2021 (link)). Briefly, pVSV-G (PT3343-5; Clontech) and psPAX2 (plasmid #12260; Addgene; http://n2t.net/addgene:12260 ; RRID:Addgene_12260; a gift from Didier Trono), together with the pCDH vector (pCDH-NLSSV40-sfCherry-NLSMyc-Blast or pCDH-sfGFP-DARPin-LA6-Hygro) in a 1:3:4 weight ratio of each plasmid was transfected into ∼80% confluent 293T cells (CRL-3216; ATCC) using Lipofectamine 3000 following the manufacturer’s instructions for lentivirus production. One day after the transfection, the medium was replaced with fresh medium, which was harvested at 48 h after transfection. For virus infection, MEFs, C2C12, BJ-5ta, and MCF10A cells were incubated with the virus-containing culture supernatants with 4 µg/ml polybrene (Nacalai Tesque) for 24 h. Infected cells were selected by incubation in medium containing 200 µg/ml hygromycin B Gold or 3 µg/ml blasticidin S (InvivoGen) for 4 d, except that C2C12 cells were selected with 20 µg/ml blasticidin S for 4 d.
5
Lentiviral Knockdown of Ask1 in RAW264.7 Cells
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Lentiviral plasmid vectors carrying Cas9 endonuclease, and sgRNA targeting Ask1 or carrying non‐targeting sgRNA, were prepared utilizing lentiCRISPRv2 plasmid (Addgene). The annealed oligos were cloned between the BsmBI restriction sites of the plasmid. The sequences of top and bottom oligos are listed in Table S1 . To produce lentivirus, HEK293T cells were transfected with Ask1‐targeting or non‐targeting lentiviral vectors, psPAX2 and pCMV‐SVS‐G (Addgene) plasmids using Lipofectamine 3000 (Thermo Fisher) in accordance with the manufacturer's protocols. To reduce cytotoxicity, the medium was replaced at 5 h after incubation. At 2 d after transfection, the supernatants containing lentivirus were harvested and filtered with 0.45 µm PVDF filter (Millipore). RAW264.7 cells were transfected with lentivirus and supplemented with 8 µg/mL polybrene (Nacalai Tesque). The medium was replaced at 1 d after transfection. Next day, the transfected cells were selected with 4 µg/mL puromycin (GIBCO).
6
Generating AT-iPS Cells via Retroviruses
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AT-iPS cells were generated according to the method as previously described28 (link). Briefly, to produce VSV-G (vesicular stomatitis virus G glycoprotein) retroviruses, 293FT cells (Invitrogen) were plated at 2 × 106 cells per 10-cm culture dish with DMEM supplemented with 10% FBS, and incubated overnight. On the next day, the cells were co-transfected with pMXs-OCT4, SOX2, KLF4 or c-MYC, pCL-GagPol, and pHCMV-VSV-G vectors using the TransIT-293 reagent (Mirus Bio LLC, Madison WI). The virus-containing supernatants were collected 48 h after incubation. The supernatants were filtered through a 0.45 μm pore-size filter, centrifuged, and then resuspended in DMEM supplemented with 4 μg/ml polybrene (Nacalai Tesque, Kyoto, Japan). Human AT1OS cells were seeded at 1.0 × 105 cells per well of 6-well plate 24 h before infection. A 1:1:1:1 mixture of OCT3/4, SOX2, KLF4, and c-MYC viruses was added to AT1OS cells28 (link)29 (link)30 (link)31 (link). The retrovirus carrying the EGFP gene was infected to estimate infection efficiency in a separate experiment. One-half of the medium was changed every day and colonies were picked up at around day 28.
7
HA-CADM1 Lentiviral Expression in HCC827 Cells
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HA‐CADM1 was cloned in a pENTR/D‐TOPO vector (Thermo Fisher Scientific). The lentiviral expression vector was then obtained by Gateway recombination with pLenti6‐V5/DEST (Thermo Fisher Scientific). The vector obtained and ViraPower Lentiviral Packaging Mix (Thermo Fisher Scientific) were co–transfected into 293FT cells using Polyethylenimine Max (Polysciences, Warrington, UK). After 48 hours, HCC827 cells were infected with the lentivirus by incubating with the culture supernatant of 293FT cells containing 5 μg/mL Polybrene (Nacalai Tesque). HCC827 cells expressing HA‐CADM1 were selected by 20 μg/mL blasticidin.
8
Retroviral Transduction of Cells
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For retroviral transduction we modified previously described methods4 (link), and used pMXs-OCT3/4, pMXs-SOX2 and pMXs-KLF4 vectors obtained from Addgene. PLAT-A packaging cells were plated at 1.2 × 106 cells per 6 cm dish, and were incubated overnight. On the following day, the cells were transfected with pMXs vectors using the Fugene HD transfection reagent (Promega). pMXs-EGFP was used as a transfection control. At 24 h after transfection, the medium was replaced with new medium, which was collected (as the virus-containing supernatant) after another 24 h. The virus-containing supernatants were filtered through a 0.45-μm pore filter and supplemented with 4 μg/ml polybrene (Nacalai Tesque). Equal amounts of supernatants containing each of the retroviruses were mixed, transferred to the cancer cell line or HUVEC dish that was prepared the previous day, and incubated overnight. At another 24 h after infection, the virus-containing medium was replaced with fresh medium without retroviruses.
9
Stable Tet3G and mNeonGreen RPE-1 Cell Lines
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RPE-1 cells stably expressing Tet3G transactivator (RPE-1 Tet3G cells) were previously described46 (link). RPE-1 Tet-On cell lines were established by retroviral-mediated integration. Each pRetroX-TRE3G plasmid and pCMV-VSV-G (Addgene, 8454) were transfected into HEK GP2-293 cells. The medium was harvested and filtered through a 0.45 μm filter (Merck Millipore, SLHVR33RS). Pre-seeded RPE-1 Tet3G cells were infected with the virus-containing medium, supplemented with fresh medium, FBS, and 4 μg/ml polybrene (Nacalai Tesque, 12996-81). RPE-1 cells stably expressing TUBB-mNeonGreen were also established by retroviral-mediated integration. pQCXIZ-TUBB-mNeonGreen and pCMV-VSV-G were transfected into HEK GP2-293 cells. The medium was harvested and filtered through a 0.45 μm filter. Pre-seeded RPE-1 cells were infected with the virus-containing medium, supplemented with fresh medium, FBS, and 4 μg/ml polybrene.
10
Cloning and Characterization of Amelx-Expressing MSCs
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MSCs were cultured in 6-cm dishes in the growth medium. When the cells reached 80% confluence, the medium was replaced with the lentiviral stock solution containing pLenti3.3/TetR, and the cells were cultured overnight at 37°C under 5% CO2 (Fig 1a ). Then, the lentivirus-containing medium was replaced with fresh growth medium. After 3 days, the cells were treated with geneticin (500 μg/ml) (Life Technologies). After 5 days, the surviving cell colonies were picked up to generate MSC clones (MSC-TetR) that strongly expressed the TetR gene. Expression of TetR in MSCs-TetR was examined by RT-PCR analysis.
MSCs-TetR were seeded at a density of 3×105 cells in a 6-cm dish in the growth medium and incubated overnight at 37°C under 5% CO2. Then, the medium was replaced with the viral stock solution of pLenti6.3/TO/V5/Amelx or plenti6.3/V5-GW/GFP supplemented with 4 μg/ml polybrene (Nacalai Tesque). After 24 hours, the cells were washed once with phosphate-buffered saline (PBS) and cultured in fresh growth medium. After 5 days, the cells were treated with 10 μg/ml blasticidin S (Funakoshi, Tokyo, Japan) to select colonies of MSCs-TetR expressing Amelx (MSCs-TetR/Amelx) or GFP (MSCs-TetR/GFP). Tet-dependent expression of the Amelx gene in MSCs-TetR/Amelx in the presence or absence of Dox (2 μg/mL) was evaluated by RT-PCR and western blotting.
MSCs-TetR were seeded at a density of 3×105 cells in a 6-cm dish in the growth medium and incubated overnight at 37°C under 5% CO2. Then, the medium was replaced with the viral stock solution of pLenti6.3/TO/V5/Amelx or plenti6.3/V5-GW/GFP supplemented with 4 μg/ml polybrene (Nacalai Tesque). After 24 hours, the cells were washed once with phosphate-buffered saline (PBS) and cultured in fresh growth medium. After 5 days, the cells were treated with 10 μg/ml blasticidin S (Funakoshi, Tokyo, Japan) to select colonies of MSCs-TetR expressing Amelx (MSCs-TetR/Amelx) or GFP (MSCs-TetR/GFP). Tet-dependent expression of the Amelx gene in MSCs-TetR/Amelx in the presence or absence of Dox (2 μg/mL) was evaluated by RT-PCR and western blotting.
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